Control of photoperiodic inhibition of luteinizing hormone secretion by dopaminergic and serotonergic systems in ovariectomized Ile-de-France ewes supplemented with oestradiol S. Le Corre and P. Chemineau INRA Physiologie de la Reproduction, Nouzilly, F-37380 Nouzilly, France The role of dopaminergic and serotonergic systems on LH secretion was investigated in Ile-de-France ewes under different artificial inhibitory photoperiodic regimens. All animals were ovariectomized at the end of the breeding season, chronically treated with an oestradiol implant, and subjected to various changes in daylength for 9 months to inhibit or stimulate their LH secretion. Plasma LH concentration was assessed by taking blood samples twice a week throughout the experiment. The effects of acute intravenous injections of the dopami- nergic2 receptor antagonist pimozide (0.08 mg kg\m=-\1) and the 5-hydroxytryptamine2 (5HT2) receptor antagonist cyproheptadine (3 mg kg\m=-\1) on LH pulsatility were assessed during challenges in four different situations: (1) long days (LD); (2) before short-day response (SD); (3) during refractoriness to short days (RSD); and (4) during inhibition by long days (ILD). LH in blood samples collected twice a week remained low during long days (0.59 \m=+-\0.03; mean \m=+-\sem), increased 45 \m=+-\1.5 days after the onset of short days and decreased 132 \m=+-\4.9 days later when ewes became refractory to short days, whereas ewes subjected to long days after 91 short days stopped their neuroendocrine activity 19 days earlier (113 \m=+-\4.7) (P < 0.01). In comparison with the pre-injection period, pimozide significantly increased the mean number of pulses in SD and RSD ewes, but not in LD and ILD ewes: SD: 0 versus 0.45 pulses in 4 h (P < 0.02); RSD: 0 versus 0.9 (P = 0.05). Cyproheptadine signifi- cantly increased the mean number of pulses in SD and RSD ewes: SD: 0 versus 1 (P < 0.008); RSD: 0 versus 1.5 (P = 0.03). An effect of cyproheptadine was shown in LD ewes (0 versus 0.5 (P < 0.03)), but it was less marked than in the same ewes under short-day photoperiod (SD ewes; P < 0.05). In ILD ewes, a small increase was observed (0 versus 0.33 (P = 0.05)) but it was less than in RSD ewes (P < 0.03). These results support the hypothesis of an inhibitory role of dopaminergic and serotonergic systems on LH pulsatile release and suggest that refractoriness to short days is due to activation of these two systems. Introduction ewes not treated with oestradiol (Meyer and Goodman, 1986; Whisnant and Goodman, 1990). However, the relative In sheep under natural photoperiod, daylength variation induces part of each of these systems in the seasonal inhibition of unknown. changes in gonadotrophin secretion that are responsible for gonadotrophin activity remains seasonal activity of reproductive function (Goodman et al, 1981). Under artificial photoperiod, transfer of ewes from a long to a Several studies have shown the role of two neural systems short photoperiod produces an increase in neuroendocrine sexual responsible for LH pulse frequency inhibition during natural activity after about 50 days. However, prolonged exposure to anoestrus. The first system is oestradiol sensitive (Legan et al, short days causes a decrease in LH pulsatility after about 140 1977) and could be mediated by catecholaminergic neurones days, making the animals photorefractory. This photorefractory since dopaminergic and adrenergic receptor antagonists state is thought to be responsible for the termination of the increased LH pulsatility in intact but not in ovariectomized breeding season in natural conditions (Robinson and Karsch, the ewes (Meyer and Goodman, 1985; 1986). The second system 1984). The neural mechanisms responsible for transduction of an have involves a direct steroid-independent influence on tonic LH photoperiodic signals into endocrine response not been release (Goodman et al, 1982) and could be controlled by sero¬ completely elucidated. It is now established that melatonin mediates the both inductive and tonergic neurones, as 5-hydroxytryptamine2 (5HT2) receptor reproductive response to inhibi¬ antagonists increase LH pulse frequency in ovariectomized tory daylengths (Bittman et al, 1983; Bittman and Karsch, 1984) and that photorefractoriness is attributed to a disruption in the "Correspondence and reprints. post-pineal processing of the photoperiodic message rather than Received 20 March 1992. to a change in the melatonin signal (Malpaux et al, 1987). Downloaded from Bioscientifica.com at 09/28/2021 07:51:53PM via free access We therefore investigated the role ot dopaminergic and ethanol-propanediol solution at a concentration or 35 mg mi \ serotonergic systems in different situations when the neuro¬ Drugs were given intravenously at a dose of 3 mg kg-1 endocrine LH activity was inhibited, in ovariectomized Ile-de- body weight. Each drug was dissolved less than 20 h before France ewes bearing subcutaneous (s.c.) oestradiol implants and intravenous injection and stored at 4°C until use. maintained under artificial photoperiodic regimens. The effects of intravenous injections of antagonists to dopaminergic2 and Drug treatments 5HT2 receptors (pimozide and cyproheptadine, respectively) on and on LH release in ewes LH pulsatility were assessed in four different situations: (1) in Effect of pimozide cyproheptadine in long days (LD); (2) before short days response (SD); (3) during long days and before short-day response. Ewes received either short days refractoriness (RSD) and (4) during inhibition by pimozide (n = 11) or cyproheptadine (n = 10) in long days long days (ILD). (LD: 19 April) and the whole procedure was repeated in the same ewes before short-day response (SD: 7 June). Control ewes received either tartaric acid (vehicle 1: = 4) or ethanol- Materials and Methods propanediol (vehicle 2: = 4). Animals and photoperiodic treatments Effect of pimozide and cyproheptadine on LH release in ewes refrac¬ tory to short days or inhibited by long days. Ewes refractory to Ue-de-France 2—9 maintained Thirty-two ewes, years old, short days or inhibited by long days received either pimozide outdoors were selected at random in December 1989. = = = They (RSD: « 4 and ILD: 6) or cyproheptadine (RSD: 5 were ovariectomized (3% halothane in for = oxygen anaesthesia) and ILD: 6). Two challenges were made (on 12 September in of the season and January at the end breeding simultaneously and 16 October). Control ewes received either tartaric acid = s.c. with a 1.5 cm Silastic diameter = = implanted implant (internal (vehicle 1) (RSD: 2 and ILD: 2) or ethanol-propanediol = 3.3 mm 4.6 et = = and external diameter mm) (Karsch al, 1973) (vehicle 2) (RSD: 2 and ILD: 2). oestradiol Chemical containing crystalline (Sigma Co., Strasbourg). During all blood were collected at and and challenges, samples Ewes were allocated according to weight age divided 20 min intervals 4 h before and 4 h after intravenous injection of into of numbers and from 2 housed two groups equal February drugs or appropriate vehicles. in a light-proof building under artificial lighting (300 lux at animal level) and subjected to two treat¬ eye photoperiodic Hormone assays ments (see Fig. la). All ewes were subjected to 90 days of long days (LD 16 h light:8 h dark; 2 February to 2 May) followed by LH was measured in a double-antibody radioimmunoassay 91 days of short days (SD 8 h light: 16 h dark; 2 May to (Pelletier et al, 1982) modified by Montgomery et al (1985). 2 August). From 2 August, 16 ewes were maintained under The sensitivity of the assay was 0.1 ng ml-1 and the intra- and short days (refractory to short days; group RSD) and 16 ewes interassay coefficients of variation were 7.0 and 9.8%, respect¬ were again subjected to long days (inhibited by long days; ively. All samples from the same challenge were measured in group ILD) until the end of the experiment (16 October 1990). the same assay. Long-term LH neuroendocrine activity LH pulse identification The effect of photoperiodic treatments on neuroendocrine LH pulses were analysed with the algorithm 'Monroe' LH activity was assessed by monitoring long-term LH vari¬ (Merriam and Watcher, 1982). The G parameters (the number of ations by collecting blood samples twice a week by venepunc¬ standard deviations by which a peak must exceed the baseline ture. Samples were immediately centrifuged for 30 min (5000 g) in order to be accepted) were 3.98, 2.4, 1.68, 1.24 and 0.93 for and plasma stored at 20°C until assay. G1-G5, respectively, for LH pulses requirements including 1 — The time of onset and cessation of LH secretion were deter¬ to 5 samples exceeding the baseline. The Baxter parameters mined arbitrarily in each ewe when the concentrations of at describing the parabolic relationship between the hormone con¬ least two consecutive LH samples were higher or below 0.8 ng centration of and the standard deviation (assay variation) of the ml-1, respectively. concentration were 0.10520 (bl, the y intercept) 0.02516 (bl, In each photoperiodic situation, drug treatments were given the coefficient) and 0.00039 (b3, the x1 coefficient). when the concentration of LH in serum of each ewe was below 0.8 ng ml-1 (three ewes were consequently excluded in each Statistical analysis drug treatment). The effects of photoperiodic treatments on weekly samples of LH were the one factor of variance Drugs compared using analysis ANOVA (photoperiodic treatments: groups RSD or ILD) with Antagonists to dopaminergic2 receptors (pimozide) and to time as a repeated measure (number of LH samples). serotonergic2 receptors (cyproheptadine) were used. Pimozide In drug treatments, the difference between mean LH concen¬ (Sigma Chemical Co., Strasbourg) was dissolved in 0.1 mmol tration after and before treatment was calculated for each ewe.